Difference between revisions of "Part:BBa K3332090"
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===Usage and Biology===
 
===Usage and Biology===
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[[File:2042.fig.1.png|none|500px|caption]]
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We use this part to be the negative control to compare with the three improved formaldehyde promoters as followed pictures by observing the fluorescence intensity/OD.
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Fig.1 Circuit
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[[File:2042.fig.1.png|none|600px|caption]]
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To construct this part, we moved formaldehyde_derivative-3 promoter (BBa_ K3332044) and RBS-ECFP-T(BBa_E0420) into the expression vector pSB1C3 by standard assembly. Then the ligation mixture was transformed into E. coli BH21 to compare the sensitivity of formaldehyde with other promoter.( formaldehyde_derivative-1 promoter, formaldehyde_derivative-2, formaldehyde promoter)
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'''Fig 1.''' Different improvements of formaldehyde promoter.
  
 
===Characterization===
 
===Characterization===
 
The agarose gel electrophoresis images are below：
 
The agarose gel electrophoresis images are below：
 
[[File:T--XMU-2090.fig.2 ger.png|none|500px|caption]]
 
[[File:T--XMU-2090.fig.2 ger.png|none|500px|caption]]
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Fig.2 pHCHO_B0034_E0020_B0015_pSB1C3 (BBa_K3332090) digested by ''Xba'' I and ''Pst'' I
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'''Fig 2.''' pHCHO_B0034_E0020_B0015_pSB1C3 (<partinfo>BBa_K3332090</partinfo>) digested by ''Xba'' I and ''Pst'' I(about 1507 bp).
  
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Protocol:  
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'''Protocol: '''
  
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1.Culture glycerol bacteria containing the corresponding plasmid in test tube for 12h.
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1.Culture glycerol bacteria containing the corresponding plasmids in test tube for 12h.
  
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2.Add 4ml of the above bacterial solution into 200 mL LB medium and maintain the culture condition at 37 ℃ and 180 rpm.
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2.Add 4 mL of the above bacterial solution into 200 mL LB medium and maintain the culture condition at 37 ℃ and 180 rpm.
  
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3.Add 0.8mM formaldehyde into each group when OD600 increased to 0.6 and the culture condition is the same as before.
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3.Add 0.8 mM formaldehyde into each group when OD<sub>600</sub> increased to 0.6 and the culture condition is the same as before.
  
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4.Then, sampling culture in 96-well plate reader every 3 hours to measure the fluorescence intensity (ECFP) and corresponding OD600, then calculate the fluorescence / OD value of each group.
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4.Then, sampling culture in 96-well plate reader every 3 hours to measure the fluorescence intensity (ECFP) and corresponding OD<sub>600</sub>, then calculate the fluorescence / OD value of each group.
  
 
Here is the result:
 
Here is the result:
 
[[File:2042.fig.4.png|none|500px|caption]]
 
[[File:2042.fig.4.png|none|500px|caption]]
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'''Fig 3.''' The curve of fluorescence intensity (ECFP) /OD by pHCHO (<partinfo>BBa_K1334002</partinfo>) and formaldehyde_derivative promoters.
  
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Fig.3 The curve of fluorescence intensity (ECFP) /OD by pHCHO (BBa_ K1334002) and formaldehyde_derivative-3 promoter
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We discovered that the three improved formaldehyde promoters are more sensitive than the registry formaldehyde promoter (<partinfo>BBa_K1334002</partinfo>). What’s more, formaldehyde_derivative-3 promoter(<partinfo>BBa_K3332044</partinfo>) is the most sensitive promoter for formaldehyde.
  
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We discovered that formaldehyde_derivative-3 promoter is more sensitive to the registry formaldehyde promoter (BBa_K1334002). What’s more, formaldehyde_derivative-3 promoter is the most sensitive promoter for formaldehyde. The detail about formaldehyde_derivative-1 promoter and formaldehyde_derivative-2 promoter can be seen in BBa_K3332042 and BBa_K3332043.
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===Sequence and Features===
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===Reference===
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[1]Yurimoto, H., Hirai, R., Matsuno, N., Yasueda, H., Kato, N. and Sakai, Y. (2005), HxlR, a member of the DUF24 protein family, is a DNA‐binding protein that acts as a positive regulator of the formaldehyde‐inducible hxlAB operon in Bacillus subtilis. Molecular Microbiology, 57: 511-519. doi:10.1111/j.1365-2958.2005.04702.x
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K3332090 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3332090 SequenceAndFeatures</partinfo>
  
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
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===Functional Parameters===
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===Functional Parameters===nfo>
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<partinfo>BBa_K3332090 parameters</partinfo>
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===Reference===
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[1]Yurimoto, H., Hirai, R., Matsuno, N., Yasueda, H., Kato, N. and Sakai, Y. (2005), HxlR, a member of the DUF24 protein family, is a DNA‐binding protein that acts as a positive regulator of the formaldehyde‐inducible hxlAB operon in Bacillus subtilis. Molecular Microbiology, 57: 511-519. doi:10.1111/j.1365-2958.2005.04702.x

Latest revision as of 22:16, 27 October 2020


Formaldehyde promoter-ECFP-terminator

It can expresse cyan fluorescent protein according to the concentration of formaldehyde.It is used to characterize the function of the improved promoter.

Usage and Biology

We use this part to be the negative control to compare with the three improved formaldehyde promoters as followed pictures by observing the fluorescence intensity/OD.

caption

Fig 1. Different improvements of formaldehyde promoter.

Characterization

The agarose gel electrophoresis images are below：

caption

Fig 2. pHCHO_B0034_E0020_B0015_pSB1C3 (BBa_K3332090) digested by Xba I and Pst I(about 1507 bp).

Protocol:

1.Culture glycerol bacteria containing the corresponding plasmids in test tube for 12h.

2.Add 4 mL of the above bacterial solution into 200 mL LB medium and maintain the culture condition at 37 ℃ and 180 rpm.

3.Add 0.8 mM formaldehyde into each group when OD600 increased to 0.6 and the culture condition is the same as before.

4.Then, sampling culture in 96-well plate reader every 3 hours to measure the fluorescence intensity (ECFP) and corresponding OD600, then calculate the fluorescence / OD value of each group.

Here is the result:

caption

Fig 3. The curve of fluorescence intensity (ECFP) /OD by pHCHO (BBa_K1334002) and formaldehyde_derivative promoters.

We discovered that the three improved formaldehyde promoters are more sensitive than the registry formaldehyde promoter (BBa_K1334002). What’s more, formaldehyde_derivative-3 promoter(BBa_K3332044) is the most sensitive promoter for formaldehyde.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Reference

[1]Yurimoto, H., Hirai, R., Matsuno, N., Yasueda, H., Kato, N. and Sakai, Y. (2005), HxlR, a member of the DUF24 protein family, is a DNA‐binding protein that acts as a positive regulator of the formaldehyde‐inducible hxlAB operon in Bacillus subtilis. Molecular Microbiology, 57: 511-519. doi:10.1111/j.1365-2958.2005.04702.x